PROJECT SUMMARY Chronic bacterial infections are difficult to treat through standard antibiotics because a small population of the bacteria becomes tolerant to antibiotics by entering a dormant state. Such bacteria are known as persisters and pose a significant problem in clinical settings. As antibiotic levels drop post-treatment, persister cells revert back into actively growing cells resulting in recalcitrant chronic infections. Currently, there are very limited approaches available for eradicating persister cells. We propose leveraging antibacterial protein colicins as an anti-persister agent. Colicins kill cells physically by disrupting cell envelopes and degrading DNA and RNA. Colicins are excellent candidates for killing persisters because the mechanisms of colicin action are growth- independent. We will utilize cell-free protein synthesis (CFPS) to produce colicins, as CFPS offers rapid protein production and characterization of such toxic proteins without cell-viability constraints. The goal of this project is to investigate and engineer colicins to control non-growing persister cells for therapeutic applications. The proposed research will be advanced with three integrated research Aims. In Aim 1, we will characterize the effects of different types of colicins on persister cells to get insights on what kind of cell killing mechanisms enable colicins to eradicate persister cells. Colicins have great potential to be developed into target-specific therapeutics without affecting beneficial bacteria. In Aim 2, we will evolve colicins via directed evolution to have enhanced activity. The engineered colicins will be identified through a fluorescence-based high-throughput screening approach using CFPS. We anticipate developing highly active and target-specific colicins that can be applied for treating persistent infections. In Aim 3, we will investigate redirecting the activity of colicins toward other pathogens which are not recognized by native colicins. The chimeric colicins will be constructed by replacing domain proteins of colicins with those of colicin-like proteins from other bacteria. Our research will open the development of customized anti-persister agents that can selectively kill antibiotic-tolerant pathogens.